Abstract The standard of care for prostate cancer (CaP) patients with localized disease requiring treatment is surgery or radiation. Those with local recurrence or metastatic CaP rely on androgen deprivation therapy (ADT). Although patients initially respond to ADT, most will become resistant to therapy through various mechanisms to reactivate androgen signaling. Research in the field has pointed to increased epigenetic plasticity as a contributor to resistance, which has resulted in the investigation of several epigenetic therapies targeting CaP. While epigenetic therapies have been successful for treatment of hematological malignancies, their success in solid tumors has been limited to preclinical studies. Their use as single agents are limited due to systemic toxicities, as the drugs target basic epigenetic enzymes used in all cell types. Combination therapies to improve target specificity are needed to reduce systemic toxicity of epigenetic therapies. Our proposal aims to take advantage of an inherent metabolic strain in CaP cells based on their high level of polyamine (PA) biosynthesis. The extraordinary level of flux through the PA pathway is driven by spermidine/spermine N1- acetyltransferase (SSAT), which utilizes acetyl-CoA to acetylate the PAs and leads to their secretion into the prostatic lumen. This strains one carbon metabolism to provide S-Adenosylmethionine (SAM) pools, which are consumed in PA biosynthesis to replenish intracellular PAs. Our current therapeutic strategy enhances stress by increasing SSAT activity combined with inhibition of the methionine salvage pathway, which recycles carbon units lost to synthesis to replenish SAM. Recent findings indicate that this metabolic disruption affects SAM and acetyl-CoA homeostasis. Our central hypothesis is that induced flux through PA metabolism and connected one carbon metabolism disrupts SAM and acetyl-CoA pool homeostasis, making CaP more sensitive to epigenetic therapy targeting SAM and acetyl-CoA utilization. Therefore, by understanding the epigenetic consequences and adaptive responses to stressed SAM and acetyl-CoA homeostasis, we can leverage this to make epigenetic therapies more efficacious at lower doses, with reduced systemic toxicity. The hypothesis will be tested by pursuing two specific aims: 1) Determine how prostate cancer cells adapt to disruption of methyl and acetyl pool homeostasis. 2) Determine how stressed PA metabolism alters the sensitivity of CaP to epigenetic therapy and whether or how this is propagated to the epigenome. We propose to combine metabolic interventions of increased polyamine catabolism and MSP inhibition with interference of histone methyltransferase or acetyltransferase activity. We expect that combination of metabolic therapies straining SAM and acetyl-CoA with epigenetic therapies targeting their usage will result in increased efficacy of epigenetic therapy and reduced systemic toxicity. .